1. Introduction
Ž .
Aquaculture of scallop Pecten maximus
larvae is often associated with high Ž
. mortality, suspected to have a bacterial etiology Nicolas et al., 1996 . Although only
Ž .
recently, a pathogen was described Lambert et al., 1998 , the bacterial etiology can be demonstrated by the prophylactic use of antibacterial agents, which increases overall
Ž .
survival of the cultures Robert et al., 1996 . So far, the agent routinely used in Ž
. European hatcheries is chloramphenicol Robert et al., 1996 . As the application of
chloramphenicol to animals intended for human consumption has now been banned in Ž
the countries of the European Union and the European Economic Area Anonymous, .
1990, 1994 , there is a need to evaluate alternative therapeutic drugs. In recent years, the antibacterial agents most used in Norwegian aquaculture have
been oxytetracycline, oxolinic acid, flumequine, florfenicol and trimethoprimrsulfadia- Ž
. Ž .
zine Tribrissenq Markestad and Grave, 1997 . The total consumption of antimicrobial
Ž agents in Norwegian aquaculture in 1997 was 555.7 kg, divided in oxolinic acid 445.5
. Ž
. Ž
. Ž
. kg , flumequine 71.4 kg , florfenicol 26.5 kg , oxytetracycline 11.8 kg and ben-
Ž .
zylpenicillinrdihydrostreptomycine 0.5 kg , measured as active component. These statistics are generated by The Department of Quality Control, Directorate of Fisheries,
Norway, on the basis of copies of veterinary prescriptions for fish. The consumption of antimicrobial agents in Norwegian aquaculture in 1987 was almost 50 000 kg, measured
as active component. The dramatic reduction observed during recent years, is due to an optimisation of the rearing conditions and development of effective vaccines.
The purpose of the present study was to establish minimum inhibitory concentration Ž
. MIC values of chloramphenicol, florfenicol, flumequine and the combination trimetho-
primrsulfadiazine for bacterial strains isolated from scallop larvae in aquaculture. Based on these MIC values, optimal therapeutic procedures for the treatment of scallop larvae
with antibacterial agents will be evaluated. Since the therapeutic procedures will be used in a marine environment, the antagonising effect of seawater on antibacterial agents was
measured.
2. Materials and methods
2.1. Source of scallop eggs and larÕae All egg groups were collected from a commercial scallop hatchery, Scalpro, Rong,
near Bergen, Norway. The broodstock originated from Hordaland County, and was conditioned in the hatchery. The two egg groups were collected in March and April
1997. The spawnings were induced by thermal shock, and eggs were fertilised by the Ž
. method described by Gruffyd and Beaumont 1970 . After fertilisation, the embryos
Ž .
were distributed in two tanks 800 l and kept in stagnant seawater at 188C 1. The seawater was obtained from the nearby fjord at 60 m and filtered using a 1 mm bag filter
Ž .
Gaf filters, Belgium . The seawater in the tanks was renewed three times a week. Ž
Larvae in one of the tanks were treated prophylactically with chloramphenicol 10 mg
y1
. l
. The first treatment was initiated 3 days following spawning and then following every renewal of water. The larvae were fed monocultures of the algae Isochrysis
Ž .
Ž .
galbana Parke Tahitian strain, PaÕloÕa lutheri Droop and Chaetoceros calcitrans Ž
.
y1
Takano 1:1:2 at a total concentration of 50 cells ml .
2.2. Isolation of bacteria Bacterial samples were taken in triplicate from one untreated tank and from one tank
treated with chloramphenicol. The total number of larvae was counted under a dissection microscope. Each sample consisted of approximately 100 000 eggs, and between 50 000
and 100 000 larvae. The eggs and larvae were washed three times in 25‰ sterile Ž
. Ž .
seawater SSW WTW, LF 196, Weilheim, Germany prior to homogenisation in 10 ml
25‰ SSW. Dilution series were made using 25‰ SSW plated out on Petri dishes with
Table 1 The strains used for MIC-testing, their day of isolation, rearing with or without chloramphenicol and
sensitivity towards diagnostic discs Or129 on blood agar for two egg groups. Strains between LT 02 and LT 28 are sampled from the first egg group, and strains between LT 43 and LT 70 are sampled from the second
egg group
Ž .
Ž .
Strain Days
Reared with q Or129
Ž .
after or without y
spawning chloramphenicol
a
LT 02 y
y LT 05
3 y
y LT 07
3 y
q LT 08
3 y
q LT 09
3 y
q LT 17
10 y
q LT 18
10 y
y LT 19
10 y
y LT 20
10 y
q LT 21
10 q
q LT 22
10 q
q LT 23
10 q
q LT 25
10 q
q LT 28
10 y
q
a b
LT 43 y
y
a b
LT 44 y
y LT 51
3 y
q LT 52
3 y
q LT 53
3 y
q
b
LT 54 10
q y
LT 58 10
q q
LT 59 10
y q
LT 60 10
y q
LT 61 10
y q
LT 62 10
y q
b
LT 63 10
y q
b
LT 70 13
y q
a
The samples were taken after fertilisation.
b
Tested on MA-plates.
Ž . Ž
. Difco 2216 Marine Agar
MA Difco, Detroit, MI, USA , Tryptone Citrate Bile
Ž . Ž
. Ž
. Sucrose agar TCBS
Oxoid, Basingstoke, England and Tryptone Soy Agar TSA Ž
. Oxoid, Basingstoke, England . Following aerobic incubation for 3 days at 188C, Colony
Ž .
Forming Units CFU were counted, and the number of colonies per larvae calculated. Representative colonies were selected for further characterisation and 14 strains from
Ž .
egg group 1 and 13 strains from egg group 2 were tested for MIC values Table 1 . All Ž
strains used in the MIC-testing were tested for Gram staining Merck, Darmstadt, .
Germany . The strains from both egg groups were tested with the diagnostic disc Or129 Ž
. 150 mg, Oxoid, Basingstoke, England . Biochemical tests were done with the commer-
Ž .
cial kit API 20E BioMerieux, France , modified by the incorporation of NaCl,
´
Ž .
according to MacDonell et al. 1982 . The kit includes the following tests: b-galacto- sidase, arginine dihydrolase lysine decarboxylase, ornithine decarboxylase, Simmon’s
Ž .
citrate, production of H S, urease Ferguson , tryptophane desaminase, indole, acetoine,
2
Ž .
protolysis of gelatin, glucose oxidative and fermentative , acidification of mannitol, Ž
. inositol, sorbitol, rhamnose, saccarose, melibiose, amygdaline and
L
q arabinose, cy- tochrome oxidase, production of nitrites, production of nitrogen, and production of gas
from glucose and catalase. In addition, some of the strains were tested for growth in ŽŽ
. different salt concentrations
TGY Tryptone Glucose Yeast, Oxoid, Baisingstoke, .
Ž .
England , and some strains were checked for the presence of flagella Leifson, 1961 . Based on the results of these characterisation tests, the isolates were classified to genus
Ž .
level according to the scheme presented by Muroga et al. 1987 . 2.3. Determination of MIC
Determination of MIC values was performed using an agar dilution method Ž
. Washington, 1985; Samuelsen and Lunestad, 1996 . The system included strains with
Table 2 Characterisation to genus level of the fermentative strains that were used in this study, according to the scheme
Ž .
by Muroga et al. 1987 Strains
Gas in glucose ‰ NaCl
VP Arginine
Lysine Genus
0.5 1
1.5 2
LT 05 y
y y
y q
q y
q y
Vibrio LT 08
y y
q q
q q
y q
y Vibrio
LT 17 y
q q
q q
q y
q y
Aeromonas LT 19
y q
q q
q q
y q
y Aeromonas
LT 20 y
y q
q q
q y
q y
Vibrio LT 22
y y
y q
q y
y q
y Vibrio
LT 23 y
y y
y q
q y
q y
Vibrio LT 28
y q
q q
q q
y q
y Aeromonas
LT 51 y
y q
q q
q y
q y
Vibrio LT 52
y y
q q
q q
y q
y Vibrio
LT 53 y
q q
q q
q y
y y
Aeromonas LT 58
y q
q q
q q
y q
y Aeromonas
LT 59 y
q q
q q
q y
q y
Aeromonas LT 60
y y
y y
q q
y q
y Vibrio
LT 61 y
y y
q q
y y
q y
Vibrio
Table 3 Characterisation of the non-fermentative strains that were used in this study, according to the scheme by
Ž .
Muroga et al. 1987 Strain
Polar Peritrichous
Genus flagellation
flagella LT 07
q y
Pseudomonas LT 09
q y
Pseudomonas LT 18
q y
Pseudomonas LT 21
y q
Agrobacterium
a a
LT 25 LT 54
q y
Pseudomonas LT 62
q y
Pseudomonas
a
Not measured.
known MIC values. For chloramphenicol and florfenicol, the reference strain was Escherichia coli ATCC 25922, whereas for flumequine and trimethoprimrsulfadiazine,
the Aeromonas salmonicida strains 7136 and 7137, respectively, from The National Veterinary Institute, Oslo, Norway, were used.
Strains from both egg groups were tested on Mueller Hinton agarrbroth supple- mented with 2 NaCl and Mueller Hinton agarrbroth made with 25‰ seawater. The
strains were transferred to 10 ml Mueller Hinton broth and incubated for 48 h at 208C, giving a final cell density of approximately 5 = 10
8
ml
y1
. Bacteria were transferred to Mueller Hinton agar using an inoculation loop of 10 ml. The Mueller Hinton agar dishes
contained increasing concentrations of the antibacterial agents: chloramphenicol, flor- Ž
.
y1
fenicol, and trimethoprimrsulfadiazine 1:5 from 0.13 to 16 mg ml in two-fold
dilution. The dishes with flumequine ranged from 0.013 to 8 mg ml
y1
in two-fold Ž
dilution. The antibacterial agents were obtained from Norsk Medisinaldepot Bergen, .
Norway . The agar dishes were incubated for 72 h at 208C. The lowest concentration of chloramphenicol, florfenicol and flumequine where complete inhibition occurred, and
the lowest concentration of trimethoprimrsulfadiazine at which marked inhibition
Ž .
Ž .
Fig. 1. Numbers of CFU in scallops cultures reared with filled symbols and without open symbols Ž
. Ž
. chloramphenicol. The measurements of CFU were performed on TCBS triangle , TSA circle and MBA
Ž .
Ž . Ž .
square agar dishes. a First egg group. b Second egg group. For the TCBS plates on D0 with and without chloramphenicol and D17 with chloramphenicol, no colonies were found.
Table 4 Ž
y1
. Summarised MIC values mg ml
for the four antibacterial agents with Mueller Hinton agar made with 2 Ž
. Ž
. NaCl NaCl or 25‰ seawater SW , for bacterial strains from both egg groups
Antibacterial agent Medium
First egg group Second egg group
Range Minimum
Range Minimum
average value average value
Chloramphenicol NaCl
0.5–16 3.2
0.13–16 2.48
SW 0.13–16
3.6 0.5– 16
9.4 Florfenicol
NaCl 0.5–8
2.8 0.13–4
1.9 SW
1–16 3.6
0.25– 16 7
Fluemequine NaCl
0.03–1 0.49
0.03–1 0.47
SW 1– 8
6.6 2– 8
6.5 Trimethoprimr
NaCl 0.5–4
1.9 0.13–4
2.01 sulfadiazine
SW 1–16
4.8 0.5–16
6.2
Table 5 Ž
y1
. Ž
MIC values mg ml for the four antibacterial agents with bacteria from both egg groups LT 02–LT 28:
. first egg group, LT 43–LT 70: second egg group tested on Mueller Hinton agar dissolved in distilled water
added 2 NaCl Strain
Chloramphenicol Florfenicol
Fluemequine Trimethoprimr
sulfadiazine LT 02
4 2
0.06 2
LT 05 0.5
0.5 0.03
0.5 LT 07
2 2
0.03 1
LT 08 1
4 1
4 LT 09
1 4
0.03 0.5
LT 17 2
4 1
2 LT 18
4 4
1 2
LT 19 2
1 1
4 LT 20
1 0.5
0.5 2
LT 21 2
2 0.5
2 LT 22
2 4
1 2
LT 23 1
2 0.5
1 LT 25
16 8
0.13 2
LT 28 2
2 0.13
1 LT 43
0.5 1
0.5 4
LT 51 2
2 1
2 LT 52
1 0.5
0.06 1
LT 53 1
4 1
4 LT 54
0.3 0.13
0.13 1
LT 58 1
0.5 0.25
2 LT 59
1 0.5
0.06 2
LT 60 2
4 1
2 LT 61
1 4
1 2
LT 62 4
2 0.5
2 LT 63
16 4
0.13 2
LT 70 0.13
0.13 0.03
0.13
occurred was recorded as the MIC value. The MIC testing was performed in duplicate. The results of the MIC-test were tested for statistical significance by the Mann–Whitney
U-test. P - 0.05 was considered significant. Ž
The pH was measured in agar plates using a pH-meter Sentron 2001, Sentron, The .
Netherlands supplied with a probe specially designed to measure pH in semisolid samples.
3. Results
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